Human Immunodeficiency Virus (HIV, HTLV-III, LAV, ARV), the cause of Acquired Immunodeficiency Syndrome (AIDS) and Aids Related Complex (ARC), differs from most known retroviruses in the complexity of its genetic organization. (For simplicity, Human Immunodeficiency Virus will hereinafter be designated as HIV. but it is understood to include all other designations which have been used in the literature to identify the virus which causes ARC and AIDS.) In addition to the Long Terminal Repeats (LTRs) and the gag, pol, and env genes present in all retroviruses, HIV has several genes which contribute to a system of genetic regulation far more complex than most retroviruses. Among these are two genes, tat and art/trs, that are both required for viral replication in vitro (A. I. Dayton et al., Cell 44, 941 (1986); A. G. Fisher et al., Nature 320, 367 (1986); and J. Sodroski et al., Nature 321, 412 (1986)). Further, the tat gene is known to code for a "transactivator" that stimulates expression of other HIV genes and the art/trs gene is believed to code for another transactivator.
Hereinafter, the genes are designated by underlining (e.g., tat and art/trs) and the gene products are designated with no underlining (e.g., tat and art/trs).
The tat gene product has been identified as a 14 kDa protein (W. C. Goh et al., J. Virology 59, 181 (1986) and M. B. Feinberg et al., Cell 46, 807 (1986)) but the putative art/trs gene product has not previously been identified.
One important viral gene regulated by tat and art/trs is the env gene which encodes the envelope glycoproteins gp120 and gp41. As a component of the viral envelope, gp120 is crucial to the interaction of the virus with its cellular receptor (J. S. McDougal et al., Science 231, 382 (1986) and P. J. Maddon et al., Cell 47, 333 (1986)) and may also contribute to the cytopathogenicity of HIV through its involvement in syncytium formation (J. Sodroski et al., Nature 322, 470 (1986). It has been reported that the degree of cytopathic effect of the virus directly correlates with the amount of viral envelope protein synthesized by an infected cell (M. B. Feinberg et al., supra).
The mechanisms by which tat and art/trs regulate env gene expression are not clearly understood. Both tat and the putative art/trs appear to act, at least in part, post-transcriptionally. In tat or art/trs defective proviral mutants there appears to be substantial viral RNA produced after proviral transfection although viral protein levels are greatly reduced (J. Sodroski et al., Nature 321, supra): M. B. Feinberg et al., supra; and C. A. Rosen et al., Nature 319, 941 (1986)) suggesting the involvement of translational control. The tat gene product may also increase the steady-state level of mRNA transcribed from the LTR although there is not yet a consensus on this point (for review see I. S. Y. Chen, Cell 47, 1 (1986)).
It has been difficult to assign precise roles to the tat and art/trs proteins in part because the two genes coding therefore overlap (in different reading frames) with each other and with the envelope gene. Studies utilizing tat or art/trs proviral deletions have been difficult to interpret due to the possibility of more than one functional unit being altered simultaneously, and the potential for other viral genes influencing the results. Further, studies that examine the effects of tat or art/trs upon the HIV LTR-directed synthesis of heterologous gene products may not be valid models if translational regulation that relies upon mRNA structure is involved.
One important aspect of research involving HIV is the development of assays to identify agents that have potential for treating AIDS victims. Such assays are designed to screen for agents that selectively interfere with the production of infectious HIV. To date, however, all such known assays are cumbersome, time consuming and require elaborate safety precautions because the assays result in the production of infectious virus. Examples of known assay systems are described in C. P. Schaffner et al., Biochem. Pharmacol. 35, 4110-4113 (1986), P. Sarin et al., N. Engl. J. Med. 313, 1289-1290 (1985), E. Terwilliger et al., J. Virol. 60, 754-760 (1986); J. Balzarini et al., Int. J. Cancer 37, 451 (1986); and H. Mitsuya and S. Broder, P.N.A.S. U.S.A. 83, 1911 (March 1986).
Two such assays look at the in vitro inhibitory effects of suramin and other various compounds which appear to inhibit activity of viral reverse transcriptase on the infectivity and replication of HIV in two test systems. J. Balzarini et al., supra. One of these assays involves following the cytopathic effect of HIV on ATH8 cells, a T-cell clone with high susceptibility to HIV, and the other of these assays involves following the expression of HIV p24 gag protein in H9 cells infected with HIV.
The ATH8 test system requires infecting the cells with HIV, culturing the infected cells in culture medium in the presence of test compound, incubating for a week or more, and then at 7-8 days after virus infection counting the number of viable cells and comparing to controls.
The H9 test system requires infecting the cells with HIV, culturing the infected cells in culture medium in the presence of test compound, incubating for a week or more, and then at days 9, 10, and 11 after virus infection determining the percentage of cells expressing the HIV p24 gag protein.
The capacity of purine and pyrimidine nucleoside derivatives to inhibit the infectivity and cytopathic effect of HIV in vitro using test systems similar to those described above has also been examined. H. Mitsuya and S. Broder, supra.
Not only are the above assays time consuming but all involve the use and expression of infectious HIV.
In view of the above, the advantages of an in vitro assay system which is relatively rapid and which does not give rise to infectious HIV are apparent.
However, the development of such an assay system has been hampered in part due to the complexity of the genetic organization of HIV as described above and also due to the fact that the putative art/trs gene product has not previously been identified.